Estrogen prevents cardiac and vascular failure in the ‘listless’ zebrafish (Danio rerio) developmental model

https://doi.org/10.1016/j.ygcen.2013.04.016Get rights and content

Highlights

  • Estrogen replacement therapy in developing zebrafish protected heart function and reduced blood vessel deterioration.

  • Removal of fluid from around the heart increases survival of aromatase inhibitor-treated fish.

  • Nitric oxide may be a critical downstream mediator of these events.

Abstract

The presence of a robust estrogen (E2) response system throughout heart and blood vessel tissues of vertebrates, including humans, has led to the speculation that this ubiquitous hormone may play a prominent role in the health and maintenance of the adult cardiovascular system (CVS). We previously established an embryonic zebrafish model called ‘listless’, which results from the inhibition of E2 synthesis by treatment with aromatase enzyme inhibitors (AI). These fish have outward symptoms similar to the human condition of congestive heart failure and tamponade. E2 replacement therapy (1) reduced the severity of cardiac sac abnormalities, (2) protected heart function, (3) prevented reduction in heart size, and (4) reduced blood vessel deterioration. Nitric oxide may be a critical downstream mediator of these events. We also demonstrate that removal of fluid around the heart increases survival of AI-treated fish. These results strongly indicate the importance of E2 in the developing CVS of the zebrafish and offer a potential model for the study of its role in CVS development, maintenance, and disease conditions.

Introduction

There is an abundance of molecular, cellular, biochemical, animal model and human patient literature to support the concept that estrogen (E2) impacts the cardiovascular system (CVS) in significant ways that may involve both genomic and non-genomic mechanisms (Turgeon et al., 2006, Duckles and Krause, 2011, Chambliss and Shaul, 2002). Indeed, a robust E2 response system exists within heart, vessel endothelial, and vessel smooth muscle cells that include E2 receptors (ERs) and the enzyme aromatase, which is responsible for the synthesis of E2 from androgens (Turgeon et al., 2006). Many prominent CVS physiological effects have also been attributed to the actions of E2 including vasodilation, anti-oxidant properties, decreased post-ischemic inflammation, and anti-atherogenesis effects (Pelligrino and Galea, 2001). A most compelling argument for a protective role of E2 in the human CVS are the numerous reports that pre-menopausal women, when compared to age-matched men or post-menopausal women, are statistically less likely to suffer from such cardiovascular diseases as stroke (Maturana et al., 2007). Indeed, CVS disease is the leading cause of death among post-menopausal women in developed countries (Maturana et al., 2007). Yet, in the face of this evidence investigators and clinicians alike were puzzled by the Women’s Health Initiative (WHI) trials involving hormone replacement therapy (HRT) that were halted before completion due to complications involving an increased risk of stroke and lack of CVS protection (Wierman and Kohrt, 2007). In response to these surprising findings, recent studies have looked more critically at the original WHI trial design and have concluded that although the data was informative, patient age, dose levels, and treatment combinations need to be reconsidered in future clinical studies. These recent studies go onto state that more basic and mechanistic E2 research needs to be pursued to better understand how to best target E2 for optimal CVS effects (Wierman and Kohrt, 2007). Most importantly, the mechanism(s) by which E2 exerts its effects on the CVS is little understood.

In the blood vasculature, non-nuclear ERα stimulates endothelial cell proliferation and migration through regulation of nitric oxide synthase (NOS) in those cells (Chambliss and Shaul, 2002). Also, E2 has been show to regulate placental angiogenesis through its regulation of VEGF and its associated growth factors (Albrecht and Pepe, 2010). In turn, investigations regarding the role of E2 in cerebrovascular protection strongly point to mechanisms involving stabilization of mitochondrial activity, suppression of vascular inflammation, and stimulation of vasodilation (Duckles and Krause, 2011). Animal model studies strongly imply that E2 affects the function of the heart. For example, studies have shown that in both pre- and post-pubertal male and female rats, gonadectomy reduces ventricular function by altering myosin isozyme patterns. However, treatment of males with testosterone and females with E2 prevents these deficiencies (Schaible et al., 1984, Scheuer et al., 1987). These observations may also apply to the role of E2 in the developing CVS.

Most importantly, the mechanism(s) by which E2 exerts its effects on the developing CVS is little understood. Therefore, there is a need to establish an in vivo developmental model in which to study the role of E2 in these CVS maturation and protective phenomena. The zebrafish is an outstanding candidate for use in E2-focused developmental studies because of its rapid maturation over several days. In addition, its transparent nature makes it ideal for observations concerning organogenesis, and drug treatments can easily be applied through its aqueous environment during early stages of maturation at concentrations similar to those used in a tissue culture paradigm (Eisen, 1996, Jesuthasan, 2002). In addition, the developmental appearance of the CVS has been well documented in the zebrafish (Lawson and Weinstein, 2002). Zebrafish also possess an active E2 response system during crucial stages of development (Callard et al., 2001, Littleton-Kearney et al., 2002). For example, aromatase is found in the zebrafish preoptic and hypothalamic regions of the central nervous system (CNS), which plays a role in sexual differentiation (Callard et al., 2001, Lassiter and Linney, 2007). Zebrafish contain two genes that code for aromatase, which convert the androgens, testosterone and androstenedione, into E2. These two genes, cyp19a1a and cyp19a1b, when transcribed and translated form the enzymes cytochrome P450 aromaA, localized in the ovary, and cytochrome P450 aromaB, localized in the brain, respectively (Callard et al., 2001). Zebrafish and goldfish, both teleosts, have a high amount of aromatase located within their brain tissue, of an order of magnitude 100–1000 fold greater than that of mammals (Callard et al., 2001). These higher levels of E2 may be the reason why teleosts such as zebrafish and goldfish so successfully regenerate damaged portions of the CNS (Callard et al., 2001).

Although E2 response systems have been found in heart and blood vessels of many species, including those of zebrafish and humans (Callard et al., 2001, Littleton-Kearney et al., 2002), no studies have used aromatase inhibitors (AIs) or selective estrogen receptor modulators (SERMs) to help identify estrogen’s role in the development of the CVS. Aromatase inhibitors (AIs) compromise aromatase enzyme activity, which is responsible for the synthesis of E2 from androgens. These inhibitors bind to the aromatase enzyme active site and prevent aromatase from catalyzing androgens into E2. For example, formestane (4-hydroxyandrostenedione or 4-OH-A) is a steroidal AI that competes with the natural substrate, androstenedione, for the enzyme and binds irreversibly to it (Harada et al., 1999). Until recently AIs have been used in zebrafish only to localize aromatase in the brain (Callard et al., 2001). SERMs were first developed with the intention of using them as antagonists to block the actions associated with ERs. However, recent findings indicate that many SERMs possess both antagonist and agonist functions (Dhandapani and Brann, 2002). SERM targets consist of any tissue that possesses ERs, which in humans include breast, uterus, bone, liver, vasculature and brain (Littleton-Kearney et al., 2002). To date the only SERM-like molecule that has been reported to not have E2 agonist activities, regarding uterine and breast cancer treatments, is the ER blocker ICI 182,780, also referred to as Faslodex, which is a newer class of drug used to treat certain types of breast cancer in postmenopausal women (Robertson, 2001). ICI is a high affinity steroidal ER antagonist, which is synthesized with the addition of an alkylamide side chain at the 7α-position (Howell et al., 2000).

Thus, AIs and SERMs appear to present ideal tools with which to help dissect out pharmacologically the role of E2 in CVS development using the zebrafish as an in vivo model. Subsequently, E2 treatment, and inhibition of E2 synthesis were initiated in recent studies to gain a better understanding of its effects on the development and maintenance of the nervous system of zebrafish (Hamad et al., 2007, Nelson et al., 2008, Houser et al., 2011). In these studies we developed an embryonic zebrafish model called ‘listless’, which was established by the inhibition of E2 synthesis through treatment with the aromatase inhibitor, 4-OH-A. The ‘listless’ fish, in addition to lacking numerous sensory-motor functions, died from CVS failure symptoms which included edema as indicated by the cardiac sac filling with fluid, gradual cessation of heart function, and collapse of blood circulation. Most significantly, these outward symptoms of CVS failure are reminiscent of congestive heart failure (CHF) in humans. These findings led to the hypothesis that E2 protects the heart and blood vessels in the developing zebrafish. The present study reports that estrogen replacement therapy (AI+E2) prevented the death of fish from both cardiac and vascular collapse. Thus, this new ‘listless’ model offers a unique opportunity to analyze the impact of E2 on CVS development and protection.

Section snippets

Zebrafish maintenance

Adult zebrafish (Danio rerio) were maintained at 28.5 °C in aquarium tanks. Fish were fed Aqueon goldfish flakes twice a day by automatic fish feeders, and freeze-dried brine shrimp at intervals throughout the week. The day-night cycle was controlled by an automatic timer, and was maintained at 14 h light-10 h dark.

Embryo collection and treatment

Wildtype embryos (strain AB) were obtained from the Zebrafish International Resource Center (University of Oregon, Eugene), the breeding facilities at Virginia Military Institute, or

AI treatment affects the embryonic heart

Treatment with the AI 4-OH-A starting at 2 dpf caused the development of edema-like symptoms throughout the embryo; the prime indicator of this phenomenon was the filling of the cardiac sac with fluid. Phase contrast micrographs (Fig. 1A and B) depict the enlarged cardiac sac of AI-treated fish compared with controls after 3 days of treatment (5 dpf). The AI-effect is dose-dependent (Fig. 1C) with both 10 and 50 μM 4-OH-A showing significant decreases at 3 dpf (1 day post treatment). Control fish had

Discussion

As we have reported in the present study, the effect of AI on the suppression of heart rate by 50% in the developing zebrafish and a return to control values over a 48 h period after its removal from the incubation medium (Nelson et al., 2008) would suggest that intrinsic E2 recovery is in some way related to cardiac function in the developing zebrafish similar to that in humans. For example, the onset of menopause and the increased incidence of cardiovascular disease are directly correlated (

Conclusions

In summary, the present study provides data suggesting that E2 is essential during the mid-to-late developmental stages of the zebrafish CVS. Heart function, ventricular size, and vascular bed integrity were severely compromised with AI administration but restored with E2 co-treatment. We also present evidence that this CVS dependence on a viable E2 response system may be related to its regulation of NOS. Indeed, there was significant constriction of the vascular bed vessels with AI treatment

Disclosure

The authors have nothing to disclose.

Acknowledgments

The authors wish to thank Ted Grigorieff, Spencer Kimori, and Chris McNair for their expert care of the zebrafish colony at VMI and Kenny Lampert for his care of the zebrafish colony at Roanoke College. This research was supported by grant funding from Virginia’s Commonwealth Health Research Board, the VMI and Roanoke College Departments of Biology, and the VMI Center for Undergraduate Research.

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